Gas chromatographic determination of deltamethrin in biological samples

Joumal of chromarograp/l_~. 246 (1982) 81-87

EIsevierscientificPublishingCompany, Amsterdam-

Printedin The Nctheriands

CHROM. 15,016

GAS CHROMATOGRAPHIC BIOLOGICAL SAMPLES*

DETERMINATION

OF DELTAMETHRIN

EN

hf. H. AKHTAR Anti& Research Centre. Research Branck &ric&ue (Received&May5th, 19S2)

Canada. Ottawa. Onzario. Kid 005

(Canada)

SUMMARY

A gas chromatographic method is described for the detection and estimation of deltamethrin in spiked samples of urine, feces, milk, liver, kidney, muscle and subcutaneous fat. It involved extraction with acetone, with the exception of milk and urine samples, liquid-liquid partition with acetonitrile, followed by clean-up on a microcolumn using benzene-hexane (1: 1) solvent system_ Milk and urine samples were extracted with hexane followed by liquid-liquid partition with acetonitrile. Under the gas chromatographic conditions employed, deltamethrin eluted in less than 4 min.

Deltamethrin,[q-a-cyano-j-phenoxybenzyl-( 1R,cis)-2,2-dimethyl-3-(2,2dibromovinyl)cyclopropane carboxylate, is one of the newly developed synthetic pyrethroids. It is also one of the most potent insecticides currently known and has a high mammalian toticity’. Synthetic pyrethroids including deltamethrin are fast gaining acceptance as agricultural pesticides_ Consequentiy, analytical techniques are needed to detect their presence in food (e.g., milk, animal tissues, etc.) and feed. Various methods, including and high-performance liquid chromatography gas chromatography (GC)2*3 have been reported for the detection and estimation of deltamethrin in (HPLC)‘s5, vegetable products_ However, at present no method is available in the literature to estimate deltamethrin residues in animal tissues. As a part of the on-going research on the metabolism of synthetic pyrethroids in livestock and poultry, analytical procedures for detection and estimation of dehamethrin in urine, feces, milk and animal tissues have heen developed and are described in this report. EXPERIMENTAL

Chemicals Labeled (“CN)

and unlabeled technical-grade

deltamethrin were prepared by

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1982 EIsevierScientilicPublishingCompany

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iU. H. _AKH-l-AR

following the published procedure 6_ In this study, the stock solution of deltarnethrin also contained smalI amounts (7-9 “/6)of [RI-z-cyano-3-phenosybnzyl-( 1R&)-2,2dinethyl-3-(2.Zdibromovinyljcyclopropane carbosyiateA sample of anal_ytical grede deltamethrin was a generous gift from Hoechst Canada Ltd. Pesticide grade solvents were used as received. Gas chrotnutographic s_r-stem A Perkin-Elmer gas chromatogaph Si_ma I, equipped with a 63Ni electroncapture detector, was used. The column used was a glass tube (1 m x 4 mm I.D.) packed with 3 ob SE-30 on SO-100 mesh Chromosorb W HP. The operating temperatures for injector, column and detector were 250, -760 and 4OO’C, respectively; methane-argon (5:95) at the rate of 35 mlimin was the carrier gas. The retention time for deltamethrin was m. 3.1 min. Under the GC conditions detailed above. 260 pg of deltamethrin gave 50” o full-scale deflection. The peak height method was used for quantitation. MeasurenIent of- raiiioactivft_3 Radicactix ity measurement of the extracts (3-O ml) was carried out on a Packard Tri-Carb liquid scintillation spectrometer Model 3320 using Bioscint (12 ml ICS. Chemical and Radioisotope Division, Irving. CA, U.S.A.), a high-efficiency emulsifier liquid scintillation cocktail_ Corrections for quenching were made. EcL’rrrction Biolo$cal samples were spiked with radiolabeied deltamethrin (106 /.LL~ = 5.2 - IO’ dDmj and its recover was calculated by both GC and radioactivity measurement techniques. _!f2k. An aliquot (15 ml) of spiked cow‘s milk was placed in a Waring blender and blended with 60 ml of hesane for 1 min. The mixture was filtered and the residue was xvashed with hesane. A known volume of ihe hexane extract (equivaIent to 10 ml of milk) was withdrawn and concentrated to ca. 3 ml on a rotary evaporator (ca. XC)_ The residue was quantitatively tranferred into a Xl-ml centrifuge tube, and the flask was rinsed with hesane (2 x 1 ml). The combined hexane layer was extracted with acetonitriIe (3 x 10 ml). and the acetonitrile phase was again shaken with hexane (2 x 10 ml) and the hesane phase was discarded_ The acetonitrile extract was dried over sodium sulfate. filtered and evaporated to dryness. The residue was redissolved in 5 ml of benzene-hesane (1 :I). An aliquot (3 ml), in duplicate, was used for radioactix-ity measurement; and 0.5 ml was subjected to column clean-up prior to GC analysis_ The detaiis on the column clean-up is described in the Clean-up technique section. Urine. Spiked cow‘s urine samples (10 ml) were diluted with 10 mi of water and shaken vigorously with 50 ml hesane in a separatory funnel. The hesane layer was collected and the aqueous phase was shaken twice with 25 ml of hexane. The combined hesane layer was dried over anhydrous sodium sulfate, fiitered and concentrat.ed on a rotary evaporator. The hexane layer (2 ml in duplicate) after appropriate volume adjustement (5 ml) was radioassayed. A portion (OS ml) of the hesane layer was anaI_yzed for deltamethrin directly on GC after necessary dilution_ Feces. An aliquot ( 10 g) of fresh feces (cow) was transferred into a wide-mouthed

GC OF DEL-I-AMETHRIN

83

erlenmeyer flask and blended with 10 ml of water and 30 ml of acetone with a Super Dispax homogenizing mill for 3 min. The mixture was filtered, and the residues including filter paper were blended twice with 10 ml water and 25 ml of acetone and re-filtered. The combined extract was transferred into a separatory funnel_ and shaken with water (100 ml) and hexane (35 ml) for 3 min. The emulsion was broken by addition of 25 ml of 2 T/,sodium chloride solution_ The hesane layer was removed and the aqueous phase was reextracted four times with 25 ml of hexane. The combined hexane extract was dried over anhydrous sodium sulfate, filtered. concentrated and the volume adjusted (5 ml). A Z-ml aliquot, in duplicate, was radioassayed. A portion (0.5 ml) was subjected to column clean-up and analyzed on GC after necessary dilution. Tksues. Fortified samples (10 g) of liver, kidney. muscle and fat tissues were homogenized with 10 ml of water and 50 ml of acetone in a Waring blender for 5 min (liver pieces were homogenized for 2 min). Prolonged homogenization with fresh liver pieces resulted in a considerable loss of deltamethrin (probably due to metabolic activity of liver enz_ymes). The homogenate was filtered and the residues were rehomogenized twice with 10 ml of water and 25 ml of acetone and filtered_ The combined filtrate was transferred to a separatory funnel ald extracted with hexane (100 ml)_ Occasional emulsions were disturbed by the addition of a sufficient amount of 29< sodium chloride colution. The hesane layer was removed and the acetonewater phase was re-extracted twice with 25 ml of hesane. The hexane estract was dried over anhydrous sodium sulfate, filtered and concentrated to a known volume (5 ml). Radioactivity of a Z-ml aliquot. in duplicate, was determined_ A 0.5-ml aliquot was subjected to clean-up on a micro-column and analyzed on a GC, after appropriate dilution_ Fatty tissues, such as subcutaneous fat samples, were difficult to homogenize_ Fat samples were mixed with washed sand (1 g of sand per gram of tissue) prior to homogenization_ The extraction and clean-up procedures were the same as detailed above. Clean-up

iechiique

The column, a disposable Pasteur pipct ( 22.9 cm), was packed with a cotton plug (pre-washed with eiuting solvent), 5 cm of deactivated Florisil(l0 Tz;water) and 3 cm of 25 % cellulose in decolorizing charcoal, in that order. The column was washed with 10 ml of benzene-hexane (1 rl) just prior to use. A known volume (0.5 ml) of the extract was placed on the column and eluted with benzene-hexane ( 1: 1). The first 3 ml were discarded. and next two 7-ml fractions were collected_ GC analyses showed that deltamethrin was completely eluted in the first 7-ml fraction; no tailing in the nest 7 ml was observed. The use of aluminum oxide (neutral) as a column packing material was also investigated_ Aluminum aside tended to retain deltamethrin longer_ and polar solvents (e.g., chloroform, dichloromethane, etc.) were required to elute the compound from the column. Identical extraction and clean-up procedures were carried out for control biological samples and the standard deltamethrin. Deltamethrin standards of known concentration (Le.. known amounts) were applied directly on the extracting solvent. and the procedures detailed above were repeated.

M_ H. XKHTAR

E-i RESULTS

AKD

DISCUSSIOS

Under the GC conditions detailed above, deltamethrin and [R]-rr-cyano-3phenoxybenzyl-( I R,cis)-2,2_dimethyl-3-( 2,24bromovinyl)cyclopropane carbosylate had the identical retention time of ca_ 3_ 1 min (2-93-L

16). The GC responses of pure

deltamethrin and the compound used in this study. which contain 7-9% of [RJ-zcyano-3- phenosybenzyl-( I R,~is)-2,2-dimethyl-3-(2,2-dibromovinyl)cyclopropane czrbosylate, were identical. The response for delramethrin was linear between 0.03 and 0.35 ns and the estimated detection limit (twice the height of the noise level) was c-a. 5 pg_ The Iimit of detection was greatly dependent on the column conditions and the oFrating temperature of the detector. In order to maintain the column at its optimnm performance, it was treated with 50 ~11of Silyl 8 after 3 days of continuous use. The detector temperature was kept at 4OO’C. A Iower detector temperature of 325 or 35O’C produced inconsistent responses after only a few injections of the biological estracts.

MILK

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TIME (MINI TIME (MIN) Fig. 1. Cbromatqrams of extracts of (a) standard deltamethrin after recovery.(b) spiked urine and (c) spiked f&es Studies were carried Qut at the 0.05ppm

lebel:

-Fig_Z_ Chromatograms of extracts of (a) standard deltamethrin after recovery. (b) spiked milk and (c) control milk. Spiking level was 0.05 ppm.

L

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GC OF DELTAMETHRIN

85

The clean-up procedure utilized in this study makes use of a liquid-liquid partition of the estracts (hexane or acetone) with acetonitrile followed by chromatography on a micro-column (Pasteur pipet) packed with deactivated Florisil (10% water) and 25 y/,cellulose in dccolorizing charcoal using benzene-hesane (1: 1) as the eluting solvent. Recovery studies were carried out on both control and spiked biological samples, and deltamethrin standards. A considerable loss (21.4-25.7 %) of deltamethrin was recorded when deltamathrin was carried through the entire extraction and cleanup procedures_ It is noteworthy that recovery of deltamethrin was excellent (915 95 %) during the clean-up, as determined by GC analyses. when applied directly on the column. Chromatograms of the extracts of the biological samples and the deltamethrin are shown in Figs. l-3. Fig. 1 records the chromatograms of (a) deltamethrin, (b) urine. (c) feces estracts of spiked samples after clean-up. Urine and feces estracts did not exhibit interfering peaks at the retention time of deltamethrin. Fig. 2 compares the chromatograms of standard deltamethrin (after recovery) with those of extracts from fortified and control milk samples Again, milk extracts did not show peaks interfering with deltamethrin. However, both control and spiked milk estracts had additional peaks close to the solvent peak. Attempts at further clean-up using hesane were not successful in removing interfering compounds_ Chromatograms of estracts of spiked and control liver, kidney, muscle and subcutaneous fat samples, and standard deltamethrin are presented in Fig. 3. Chromatograms of the control biological samples did not exhibit any peaks that interfered with deltamethrin. This helped the estimation of deltamethrin in the spiked samples_ Recovery of deltamethrin from spiked biological samples are shown in Table I. The data are based on the assumption that both deltamethrin and the other isomer (7-9 %) behaved identically during the estraction and clean-up procedures. In addition, data in Table I take into account the losses of deltamethrin during the estraction and clean-up steps. The values in Table I were generated by comparing the recovery values of deltamethrin from biological samples with those obtained without biological samples. In other words, the recovery values of deltamethrin without biological samples have been treated as a standard (100%) and the other values are relative to these values and are corrected values. Data in Table I are based on both radioactivity measurement and GC analyses of the extracts. Radioactivity data were recorded prior to clean-up, and in most cases were seater than 90%. On the other hand, GC data were obtained after clean-up, with the esception of urine_ and the recovery was in the 80% range_ The differences between the radioactivity and GC recovery data suggest that a portion of deltamethrin, although extractable, is somehow bound to biological materials and is being retained on the column during the column clean-up. GC analyses of estracts of spiked liver samples (fresh) show considerable loss of deltamethrin, yet the radioactivity data are still in 90% range. It may be due to

transformation

of deltamethrin

by the action of liver enzyme(s) into product(s) con-

taining lJCN moiety. This phenomenon would reduce the concentration of deltamethrin while maintaining the radioactivity counts The effect of liver enzyme(s) on the fate of deltamethrin is being studied further_ The method detailed above permits extraction, isolation and quantitation of

M. H. AKHTAR

86

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LIVER

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2 TIME

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Chromatograms of extracts. (I) Liver:

TIME

(MIN

)

(a) deltamethin; (b) spiked liver; (c) control liver- (iii Kidney: {a) deltamethrin; (5) spiked kidney; (c) control kidney. (iii) Fat: (a) dehmethrin; (b) spiked fat; (c)control 51:. (iv)hIuscie:(a)ddtamethrin; (b)spiked musde;(c)cxmtrol sample_Oeltamethrinrefersto st~dYddeitamethrin~erraov~_and all thestudieswerc conductezdattheO.l-ppmIew9. Fig_ 3.

CC OF DELTAMETHRIN TABLE

57

I

RECOVERY

Sample

OF DELTAMETHRIN

Added

Uecovev

FROM

BIOLOGICAL

SAMPLES

(%)*m-

IPP4

GC method Urine

0.1 0.05 0.01

Feces

0.1 0.05

98-104 51-87 76-8 1

Radioactivity 99-102 95-97 93-97

7%79

95-98

0.01

x-75 67-73

89-93 93-94

Milk

0.1 0.05 0.01

83-87 SO-89 67-72

99-102 96-97 93-94

Liver

0.1 0.05 0.01

83-89 67-75 68-73

92-94 93-95 78-81

Kidney

0.1 0.05 0.01

82-9 1 81-87 70-78

93-102 87-89 93-94

lMuscle

0.1 0.01

88-95 83-94

S8-89 93-95

Fat

0.1 0.01

SO-!?6 75-79

102-104 93-103

l For three determinations each. t* Corrected using deltamethrin recovery values without biological samples as a standard.

detlamethrin from bioiogical samples. The techniques detailed here are being used in in vitro and in viw studies of deltamethrin with poultry and farm animals. These procedures are also being extended to include the extract residues of cypermethrin and fenvalerate from biological samples. ACKNOWLEDGEMENT

The author acknowledges the technical assistance of Claude Danis with this study. REFERENCES M_ Elliot, N. F_ Jams and C. Potter, Am. Rev. firomol., 23 (1978) 443-469. R ~Uestres,C. ChevalIier, C. Fspinoza and R Cornet, Tram. Sot. Pharm. Montpeilier,

38 (1978) 183191; CA., 90 (1979) 1SIk M_ Hasmet 2nd IL Lab-e, Ph4r~tr_-Ph_~top~rm_, _97 (1978) 85-97; CA. 90 (1979) 181517a. 1. Chromfogr.. 176 (1979) 140-144. C_ lUeinh& J. C. Suglia and P. B runea, D. Mourot, D. D&pine, J. Bokseau and G. Gayot, J_ Chronzurogr., M. Elliot, A_ W. Fanhun, N. F_ James, P_ H. N&ham and D. A (1974) 710.

173 (1979) 412-414. Puhan. Nature (London/.

248